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Plasmid Files

pMT-DEST48

Gateway® destination vector for inducible high-level expression of C-terminally V5-6xHis-tagged proteins in Drosophila cells.

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pMT-DEST48 Sequence and MappMT-DEST48.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 EcoO109I (5169) AatII (5115) ZraI (5113) NmeAIII (4341) AhdI (4193) PspFI (3608) BseYI (3604) PciI (3300) BspQI - SapI (3184) SbfI (2933) MfeI (2824) HpaI (2815) PsiI (2795) PmeI (2646) 6xHis AgeI (2616) MluI (2610) PfoI (46) BstAPI (185) KasI (235) NarI (236) SfoI (237) PluTI (239) BsgI (634) NotI (979) BspEI (1295) EcoRI (1299) PasI - PflMI * (1531) BtgI - NcoI - StyI (1600) BssHII (1793) BstZ17I (1834) BbvCI (2024) AvaI - BsoBI - TspMI - XmaI (2168) BmeT110I (2169) SmaI - SrfI (2170) BstXI (2287) BstBI (2566) BseRI (2583) pMT-DEST48 5179 bp
EcoO109I  (5169)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
AatII  (5115)
1 site
G A C G T C C T G C A G
ZraI  (5113)
1 site
G A C G T C C T G C A G
NmeAIII  (4341)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII
recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
AhdI  (4193)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
PspFI  (3608)
1 site
C C C A G C G G G T C G
BseYI  (3604)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its
electrophoretic mobility.
PciI  (3300)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3184)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (3184)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be
mixed before removing an aliquot.
SbfI  (2933)
1 site
C C T G C A G G G G A C G T C C
MfeI  (2824)
1 site
C A A T T G G T T A A C
HpaI  (2815)
1 site
G T T A A C C A A T T G
PsiI  (2795)
1 site
T T A T A A A A T A T T
PmeI  (2646)
1 site
G T T T A A A C C A A A T T T G
AgeI  (2616)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
MluI  (2610)
1 site
A C G C G T T G C G C A
PfoI  (46)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
BstAPI  (185)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
KasI  (235)
1 site
G G C G C C C C G C G G
NarI  (236)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
SfoI  (237)
1 site
G G C G C C C C G C G G
PluTI  (239)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
BsgI  (634)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
NotI  (979)
1 site
G C G G C C G C C G C C G G C G
BspEI  (1295)
1 site
T C C G G A A G G C C T
EcoRI  (1299)
1 site
G A A T T C C T T A A G
PasI  (1531)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
PflMI  (1531)
1 site
C C A N N N N N T G G G G T N N N N N A C C
* Blocked by Dcm methylation.
Sticky ends from different PflMI sites may not be compatible.
BtgI  (1600)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
NcoI  (1600)
1 site
C C A T G G G G T A C C
StyI  (1600)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
BssHII  (1793)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
BstZ17I  (1834)
1 site
G T A T A C C A T A T G
BbvCI  (2024)
1 site
C C T C A G C G G A G T C G
AvaI  (2168)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (2168)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
TspMI  (2168)
1 site
C C C G G G G G G C C C
XmaI  (2168)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
BmeT110I  (2169)
1 site
C Y C G R G G R G C Y C
SmaI  (2170)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (2170)
1 site
G C C C G G G C C G G G C C C G
BstXI  (2287)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
BstBI  (2566)
1 site
T T C G A A A A G C T T
BseRI  (2583)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the
DNA.
AmpR
4120 .. 4980  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4120 .. 4911  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4120 .. 4980  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4912 .. 4980  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4120 .. 4980  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
CmR
1086 .. 1745  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1086 .. 1745  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
3361 .. 3949  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3361 .. 3949  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
MT promoter
401 .. 827  =  427 bp
Drosophila metallothionein promoter
MT promoter
401 .. 827  =  427 bp
Drosophila metallothionein promoter
ccdB
2087 .. 2392  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
2087 .. 2392  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
SV40 poly(A) signal
2681 .. 2815  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2681 .. 2815  =  135 bp
SV40 polyadenylation signal
attR1
853 .. 977  =  125 bp
recombination site for the Gateway® LR reaction
attR1
853 .. 977  =  125 bp
recombination site for the Gateway® LR reaction
attR2
2433 .. 2557  =  125 bp
recombination site for the Gateway® LR reaction
attR2
2433 .. 2557  =  125 bp
recombination site for the Gateway® LR reaction
AmpR promoter
4981 .. 5085  =  105 bp
AmpR promoter
4981 .. 5085  =  105 bp
V5 tag
2571 .. 2612  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
2571 .. 2612  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
lac UV5 promoter
1002 .. 1032  =  31 bp
   Segment 1:  -35  
   1002 .. 1007  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1002 .. 1032  =  31 bp
   Segment 2:  
   1008 .. 1025  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1002 .. 1032  =  31 bp
   Segment 3:  -10  
   1026 .. 1032  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1002 .. 1032  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
6xHis
2622 .. 2639  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2622 .. 2639  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
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